CN114487059B - Electrochemical luminescence immunosensor and preparation and application thereof - Google Patents

Electrochemical luminescence immunosensor and preparation and application thereof Download PDF

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CN114487059B
CN114487059B CN202111646274.4A CN202111646274A CN114487059B CN 114487059 B CN114487059 B CN 114487059B CN 202111646274 A CN202111646274 A CN 202111646274A CN 114487059 B CN114487059 B CN 114487059B
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CN114487059A (en
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李建国
马国雨
张孙小艺
吴康
邓安平
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Suzhou University
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The invention belongs to the field of electrochemical sensors, and particularly relates to an electrochemical luminescence immunosensor and preparation and application thereof. Discloses a method based on MoS 2 A competitive electrochemical luminescence immunoassay method for detecting pentafluorouracil with an ultra-sensitivity of a @ GO substrate and a Ru-MOF luminescent probe and a detection application in a human serum sample. The invention prepares Ru-MOF nanosheet and MoS 2 The @ GO nano sheet is respectively used as a luminescent probe and a modified electrode substrate, and an electrochemical luminescence immunosensing method for detecting the pentafluorouracil with ultrasensitivity is developed. The immune sensor uses simple instruments and equipment, is simple and convenient to operate, has high detection efficiency, simple sample treatment, high sensitivity and wide linear range, is convenient to popularize, has lower cost and has high practical value.

Description

Electrochemical luminescence immunosensor and preparation and application thereof
Technical Field
The invention belongs to the field of electrochemical sensors, and particularly relates to an electrochemical luminescence immunosensor and preparation and application thereof.
Background
In the actual clinical treatment of malignant tumors (including head and neck and gastrointestinal tumors) around the world, fluoropyrimidine is the basic drug currently used in the chemotherapy technology, wherein pentafluorouracil (5-fluorouracil, 5-FU) is the chemotherapy drug with the first three usage rates. Meanwhile, because of the radiosensitivity of the fluoropyrimidine, the fluoropyrimidine is often combined with external beam radiotherapy in the actual clinical treatment process. As with other chemotherapeutic agents, the risk of fluoropyrimidine use and its drug-related toxicity are not negligible and must be weighed against their potential benefits by a multi-fold compromise. 5-FU has severe side effects in use, such as alopecia, erythema dermatitis, skin pigmentation, hand-foot syndrome, and transient cerebellar movement disorder. Meanwhile, according to the existing clinical data, 5-FU is a common medicine related to heart diseases, which is ranked second only to anthracycline medicines, and the most common cardiac toxicity related to fluoropyrimidine is chest pain (atypical chest pain, angina pectoris during exercise or rest) and acute coronary syndromes including myocardial infarction; other less common cardiotoxic manifestations include atrial fibrillation, arrhythmia, myocarditis and pericarditis, heart failure and even death. Although the cardiotoxicity associated with fluoropyrimidines remains an undefined entity, the necessity for a definitive link between the two remains to be explored further. But the life is never taken a little bit, and the side effects and the potential risks which are already generated remind us that more caution is needed for the clinical use of the medicine.
So far, no well-established and complete mechanism has been formed for the real-time monitoring of 5-FU concentration, but a number of previous studies have shown that the application of the real-time monitoring of 5-FU concentration in clinical medicine is a promising candidate. For example, in 2015, shikandard, bukkitgar and Nagaraj pMethylene blue was deposited and a novel sensor for the determination of 5-FU was established, which has been successfully applied in pharmaceutical and biological fluid sample analysis. In 2021, a new COF hybrid material EB-TFP was elaborately designed by the king group: eu (BTA) 4 As a sensing platform for the proportional fluorescence detection of 5-FU, the strategy realizes quantitative and selective fluorescence response of 5-FU.
Electrochemiluminescence (ECL) has many advantages over Chemiluminescence (CL), photoluminescence (PL), and electrochemical methods. Compared with the CL method, the ECL method has stronger controllability and selectivity; compared to the PL method, the ECL method does not require a light source; the ECL process has greater selectivity and less electrode contamination than the electrochemical process. Therefore, the ECL not only solves the problems such as scattered light and luminescent impurities, but also has a wide application in the fields of analytical chemistry and biosensing due to its advantages of good applicability, simple instrument, low background signal, wide linear working range, high sensitivity, etc. At present, ECL becomes a mature analysis technology and is widely applied to the fields of clinical examination, immunoassay, food and water quality detection, biological warfare agent detection, drug analysis and the like.
Immunoassays are powerful analytical methods based on the high specificity of antigen and antibody reactions, which form the basis of Radioimmunoassays (RIA). Currently, this assay has been successful in screening for the presence, progression and treatment outcome of a disease or infection by the use of molecular markers; it also has application in monitoring the presence and changes of drugs, toxic substances and environmental pollutants. The mainstream immunoassay methods so far mainly include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrophoretic Immunoassay (EIA), fluorescent Immunoassay (FIA), chemiluminescent immunoassay (CLIA), electrochemical immunoassay (ECIA), electrochemical luminescent immunoassay (ECLIA), and the like. However, ECLIA has the advantages of high selectivity, high sensitivity, simple equipment, low background signal, no radioactive or toxic label, and wide detection range, and thus, in recent years, the detection method has attracted great attention of researchers all over the world. ECLIA primarily includes both sandwich-type and competitive-type assays. In sandwich immunoassays, detection is generally performed by ECL luminescence signals using primary antibodies (Ab 1) immobilized on the electrode surface as capture probes, and then binding to target antigens and ECL reagent-labeled secondary antibodies (Ab 2) as signal probes. In a competitive immunoassay, an unlabeled analyte (usually an antigen) in a test sample is measured by its competing reaction with a labeled antigen in the immunoassay. Unlabeled antigens hinder the ability of labeled antigens to bind because the binding sites on the antibody are already occupied.
Disclosure of Invention
The invention aims to solve the defects of the existing detection method of the pentafluorouracil, and aims to construct an ECL (immuno-sensing) method based on Ru-MOF (metallothionein), which can be applied to detecting the content of 5-FU in blood or urine of a cancer patient and provide reliable data for clinical medicine as a reference for a doctor to further formulate a treatment scheme.
The invention provides a preparation method of an electrochemiluminescence immunosensor, which comprises the following steps:
(1) Mixing zinc nitrate hexahydrate, polyvinylpyrrolidone, ru (dcbpy) 3 2+ Dissolving ruthenium chloride CAS (4,4-dicarboxydipyridine) 97333-46-5) and pyrazine in water, reacting and purifying to obtain a Ru-MOF solution;
will be (NH) 4 ) 6 Mo 7 O 24 Dissolving in graphene oxide dispersion, keeping at 40-60 deg.C for 4-6h, adding thiourea, heating, removing impurities to obtain MoS 2 @ GO solution;
(2) Activating the MoS 2 In the @ GO solution and the Ru-MOF solution, removing impurities to respectively obtain MoS 2 A @ GO activation solution and a Ru-MOF activation solution;
(3) Adding a 5-FU antibody solution into the Ru-MOF activation solution, and reacting to obtain a Ru-MOF/Ab probe;
(4) The MoS is processed 2 The @ GO activation solution is dropwise added on the surface of a glassy carbon electrode, the dried glassy carbon electrode is dropwise added with a 5-FU coated antigen solution for incubation, a BSA solution is added, and impurities are removed to obtain MoS 2 @ GO/OVA substrates;
the Ru-Dropping MOF/Ab probes to the MoS 2 And reacting on the @ GO/OVA substrate to obtain the electrochemical luminescence immunosensor.
Preferably, in the step (1), the reaction temperature is 70-90 ℃ and the reaction time is 20-30h.
Preferably, the preparation method of the graphene oxide dispersion liquid is to mix graphite, potassium peroxodisulfate and phosphorus pentoxide in concentrated sulfuric acid, and heat to obtain a mixed solid; and (3) reacting the mixed solid with sulfuric acid and potassium permanganate, dispersing in water, and removing impurities to obtain a graphene oxide dispersion liquid.
Preferably, in the step (1), the heating condition is that the temperature is kept at 40-60 ℃ for 0.8-1.2h, and then the temperature is increased to 180-220 ℃ for 20-28h.
Preferably, in the step (1), (NH) 4 ) 6 Mo 7 O 24 The temperature for reacting with the graphene oxide dispersion liquid is 40-60 ℃, and the reaction time is 4-6h; the thiourea is added for reaction, the reaction is carried out for 0.8 to 1.2 hours at the temperature of between 40 and 60 ℃, and the reaction is carried out for 20 to 28 hours after the temperature is increased to between 180 and 220 ℃.
Preferably, in the step (1), the (NH) 4 ) 6 Mo 7 O 24 And thiourea in a molar ratio of 1:16-25.
Preferably, in the step (2), the activation method is to add a NHS/EDC solution, and the activation time is 1-2h; the concentration of the NHS/EDC solution is 7-9mg/mL.
Preferably, the volume ratio of the Ru-MOF to the 5-FU antibody is 1:0.5-2.
Further, the impurity removal mode is to dissolve after centrifugation.
Further, in the step (3), a BSA solution is added before the Ru-MOF activation solution and the 5-FU antibody solution react; the volume ratio of the BSA solution to the Ru-MOF/Ab probe is 1:0.5 to 2; the concentration of the BSA solution is 4-6wt%; the reaction temperature is 3-5 ℃, and the reaction time is 1-2h.
Further, in the step (4), moS is activated 2 The mass ratio of the @ GO to the 5-FU coating antigen is 1:15-25; the incubation temperature is 3-5 ℃, and the incubation time is 8-16h.
The invention also provides the electrochemiluminescence immunosensor prepared by the preparation method.
The invention also provides an application of the electrochemiluminescence immunosensor in 5-FU detection, which comprises the following steps:
(1) Performing cyclic voltammetry scanning on a 5-FU standard sample in a three-electrode sensing mode, drawing a luminous intensity-concentration curve, and establishing a linear relation to obtain a linear regression equation; the electrochemical luminescence immunosensor is used as a working electrode, a platinum electrode is used as an auxiliary electrode, ag/AgCl is used as a reference electrode, and a PBS solution containing potassium peroxodisulfate is used as a buffer solution; electrochemical conditions were set as: in the electrochemical window range of-1.6-0V, carrying out cyclic voltammetry scanning on a photomultiplier at a high voltage of 700V and a scanning speed of 0.2V/s;
(2) Carrying out ECL detection on the treated sample to obtain the luminous intensity; and calculating the luminous intensity by using the linear regression equation to obtain the concentration of the pentafluorouracil in the sample.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention aims to provide an ECL immunosensing method based on Ru-MOF (ruthenium-metal-organic framework) aiming at the defects of the existing detection method of the pentafluorouracil, which can be applied to detecting the content of 5-FU in blood or urine of a cancer patient and provide reliable data for clinical medicine as a reference for doctors to further formulate a treatment scheme. Compared with the existing 5-FU detection method, the immunosensing method has the advantages of strong controllability in time, space and luminous intensity, simple operation, cost benefit, high sensitivity, low background signal and low reagent consumption.
Drawings
FIG. 1 is a schematic diagram of the construction and detection mechanism of an immunosensor;
FIG. 2 is a graph of ECL intensity vs. concentration of (A) various concentrations of pentafluorouracil: the concentration of 5-FU is (a) 0.0001ngmL -1 ,(b)0.001ng mL -1 ,(c)0.01ng mL -1 ,(d)0.1ngmL -1 ,(e)1ng mL -1 ,(f)10ng mL -1 And (g) 100ng mL -1 (ii) a (B) Adding linear regression curve graphs of the luminous intensity values of the pentafluorouracil with different concentrations and the logarithm of the concentration of the pentafluorouracil;
FIG. 3 is (A) a TEM image of Ru-MOF nanoplates; (B) a fluorescence emission spectrum of Ru-MOF nanosheets;
FIG. 4 shows (A) MoS 2 TEM images of @ GO nanosheets; (B) MoS 2 The EDS spectrogram of @ GO nanosheet.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
In the following examples of the present invention, 5-FU-coated antigen and 5-FU monoclonal antibody were given to the subject group of professor Deng Anping, university of Suzhou. Wherein the 5-FU monoclonal antibody is a monoclonal antibody secreted by a 5-FU hybridoma cell line 4G11-A11 (preserved in China center for type culture Collection with the preservation number of CCTCC No. C20220249 and the preservation address of Wuhan university in eight-channel Hongshan district, wuhan City, hubei province). The preparation of the antibody comprises the steps of firstly determining a proper modification site in a 5-FU molecular structure according to a hapten molecular design theory and 5-FU molecular structure characteristics, synthesizing two 5-FU modifiers with different connecting arm lengths and carboxyl end groups, connecting the 5-FU modifiers with carrier protein, and then preparing the high-sensitivity and high-specificity anti-5-FU monoclonal antibody through the steps of animal immunization, fusion, screening and the like by applying a hybridoma antibody preparation technology. IC of ELISA method for determining 5-FU established based on antibody 50 The value is 20ng/mL, and the cross reaction rate of the compound with uracil, cytosine, thymine, 5-bromouracil, 5-fluoro-1,3-dimethylpyrimidine, uridine, 5-bromo-2' -deoxyuridine, capecitabine and gimeracil is less than 0.1 percent.
Example 1
The synthesis steps of Ru-MOF are as follows: 9.0mg of Zn (NO) 3 ) 2 ·6H 2 O、20mg PVP(40000)、9.0mg Ru(dcbpy) 3 2+ And 1.6mg pyrazine into a 50mL three-necked round bottom flask, 32mLH was added 2 Dissolving it in O, and ultrasonic treating the solution 5min to disperse it uniformly. The mixture is stirred slightly at 80 ℃ and reacted for 24 hours. After completion of the reaction, the obtained suspension was centrifuged (12000rpm, 10 min) to obtain orange crystals, and after removing the supernatant, the precipitate was washed 3 times with deionized water. Finally, the washed orange crystalline product was redissolved in 8mLH 2 In O, the mixture is dispersed evenly by ultrasonic treatment for 5min and stored in an environment of 4 ℃ for further use.
Preparation of Graphene Oxide (GO): 2g of graphite, 2g K 2 S 2 O 8 And 2g P 2 O 5 At 15mL,98% of 2 SO 4 After mixing, and heating at 80 ℃ for 10h. The solid obtained is then placed in H at 40 ℃ 2 SO 4 6g of KMnO 4 And (5) oxidizing for 2h. After preparation, GO was kept well for use, and before use, GO was dispersed in water and the suspension was dialyzed for two weeks.
MoS 2 Preparation of @ GO: 2.328g (NH) 4 ) 6 Mo 7 O 24 Dissolve in 30mL of 1mg/mL GO water solution, mix well, keep the reaction at 50 ℃ for 5h, then dissolve 3.197g thiourea in the solution and stir the reaction at 50 ℃ for 1h. Finally transferring the prepared precursor into a Teflon high-pressure reaction kettle, keeping the reaction kettle at the temperature of 200 ℃ for 24 hours, centrifuging the suspension after the reaction is finished to obtain a black solid, removing the excessive thiourea solution on the upper layer, washing the black solid with ethanol and ultrapure water for 3 times, and finally freezing and drying the black solid to obtain MoS 2 @ GO, which is preserved properly for use.
Activation of Ru-MOF: first, 50. Mu.L of the prepared Ru-MOF was taken and 100. Mu.LH was added 2 Diluting with O, performing ultrasonic treatment for 2min to uniformly disperse, adding NHS and EDC into the solution to activate carboxyl, and standing for 1h at 4 ℃. Finally, centrifuging the activated Ru-MOF solution to obtain a trace amount of orange crystals, and then using 150 mu LH 2 And O, redissolving the mixture for later use.
Preparation of Ru-MOF/Ab Probe: an antibody (Ab) to 5-FU is attached to Ru-MOF through an amide bond (-CO-NH-) to form a Ru-MOF/Ab probe. 10 mu LRu-MOF and 10 mu L5-FU antibody are added into a centrifuge tube, mixed evenly and placed in an environment at 4 DEG COvernight (about 12 h). After the reaction was completed, 10. Mu.L of 5% BSA (bovine serum albumin) was added dropwise to the centrifuge tube, the tube was left at 4 ℃ for 1 hour to block the nonspecific binding sites and to avoid nonspecific binding, and finally, the tube was centrifuged and then 10. Mu. L H was added 2 O or different concentrations (0.0001 ng mL) -1 ,0.001ng mL -1 ,0.01ng mL -1 ,0.1ng mL -1 ,1ng mL -1 ,10ngmL -1 ,100ngmL -1 ) And re-dissolving the 5-FU standard solution to obtain the Ru-MOF/Ab probe.
Example 2
Preparing an electrode: the GCE (glassy carbon electrode) is coated with alpha-Al on chamois 2 O 3 Polishing to be mirror-surface-shaped, washing the polishing powder with ethanol and ultrapure water, drying with nitrogen, and standing aside for later use.
MoS 2 Activation of @ GO: firstly, the prepared MoS is prepared 2 After @ GO is diluted to the target concentration, adding a proper amount of NHS and EDC into the solution to activate carboxyl, and then standing the solution for 1h in a room temperature environment. Finally, the activated MoS is treated 2 Centrifuging the @ GO solution to obtain a small amount of black solid, and adding a proper amount of H 2 And O, redissolving the mixture into a suspension with a target concentration for later use.
Modifying the electrode: take 10 mu LMoS 2 And (3) dropwise adding the @ GO suspension to the surface of a clean electrode, and adding a proper amount of dry beads for drying. After the electrode surface was dried, 10. Mu.L of 5-FU-coated antigen was dropped on the modified electrode, and then left to incubate overnight (about 12 h) in an environment at 4 ℃. Finally, 10. Mu.L of 5% BSA (bovine serum albumin) was dropped on the surface of the modified electrode, and after the electrode was left to stand at 4 ℃ and blocked for 1 hour (blocking of non-specific binding sites), the unbound 5-FU-coated antigen and BSA were washed away with 0.01M PBS, whereby MoS was obtained 2 @ GO/OVA substrates.
Assembling the immunosensor: the prepared Ru-MOF/Ab probe (10. Mu.L) was added dropwise to MoS 2 The @ GO/OVA substrate surface is placed in an environment at 4 ℃ for reaction for 1h, and finally the MoS which is not reacted with the GO is washed away by 0.01M PBS 2 The assembly of the immunosensor is completed by the Ru-MOF/Ab probe connected with the substrate of @ GO/OVA.
Example 3
The assembled electrochemiluminescence sensor is used as a working electrode, a platinum electrode is used as an auxiliary electrode, ag/AgCl is used as a reference electrode to form a three-electrode system, and PBS (pH 7.4) containing 0.1M potassium peroxodisulfate is used as ECL detection buffer solution. In the electrochemical window range of-1.6-0V, the high voltage (PMT) of the photomultiplier tube is 700V, the sweep rate is 0.2V/s, and cyclic voltammetry scanning is carried out. Measuring ECL signals of a series of 5-FU standard solutions (0.0001 ng/mL-100 ng/mL) with different concentrations, measuring and recording a luminous intensity-5-FU concentration curve, establishing a linear relation between ECL luminous intensity and a pentafluorouracil concentration logarithm value according to measured data, wherein a regression equation is as follows: i =5966.9-1373.0lg c 5-FU Coefficient of regression R 2 =0.998, the detection limit is approximately 0.03pg/mL.
TABLE 1ECL immunosensor for spiking recovery data of pentafluorouracil in real samples
Figure BDA0003443951690000061
N.D.＝not detected
And (3) treating the collected serum sample, adding water (blank sample) or the pentafluorouracil with different concentrations to perform a labeling experiment, performing ECL test, obtaining the luminous intensity, and calculating the obtained luminous intensity by using the obtained linear regression equation to obtain the concentration of the pentafluorouracil in the sample. The results are shown in table 1, the recovery rate of the sensor to the pentafluorouracil in the standard sample is in a range of 86.7% -92.8%, and the RSD is in a range of 5.9% -10.4% (n = 3), which indicates that the strategy can be used for detecting the pentafluorouracil in actual samples more accurately.
Effect evaluation 1
The work aims to construct a competitive ECL immunosensing method based on Ru-MOF, can be applied to detecting the content of 5-FU in blood of a cancer patient, provides reliable data for clinical medicine, and is used as a reference for doctors to further formulate a treatment scheme. Ru-MOF/Ab was used as a probe in this work because tris (4,4 '-dicarboxylic acid 2,2' -bipyridyl) -ruthenium (II) (Ru (dcbpy) 3 2+ ) Good water solubility andexcellent ECL properties, making it possible for ligands to form functionalized metal-organic frameworks (MOFs) with MOF nanoplates (Ru-MOFs). In addition, moS was used in this experiment 2 @ GO as a substrate to modify electrodes, moS 2 The @ GO can form a thin two-dimensional nano material modification layer, so that the electron transfer rate is improved, and meanwhile, more 5-FU coated antigen can be loaded due to the large specific surface area of the @ GO. The work not only provides a new idea for detecting 5-FU and other drug molecules, but also widens the application range of the ECL immunosensor.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims (8)

1. The application of the electrochemiluminescence immunosensor in 5-FU detection is characterized by comprising the following steps:
s1: in a three-electrode system, the electrochemical luminescence immunosensor is used as a working electrode, cyclic voltammetry scanning is carried out on a 5-FU standard sample, a luminescence intensity-concentration curve is drawn, and a linear regression equation is obtained;
s2: performing ECL detection on the treated sample to obtain the luminous intensity; calculating the luminous intensity by using the linear regression equation to obtain the concentration of the pentafluorouracil in the sample;
the electrochemical luminescence immunosensor is prepared by the following method:
(1) Mixing zinc nitrate hexahydrate, polyvinylpyrrolidone, ru (dcbpy) 3 2+ Dissolving pyrazine in water, and reacting to obtain a Ru-MOF solution;
will be (NH) 4 ) 6 Mo 7 O 24 Dissolving in graphene oxide dispersion, maintaining at 40-60 deg.C for 4-6h, adding thiourea, and heating to obtain MoS 2 @ GO solution;
(2) Activating the MoS 2 In the @ GO solution and the Ru-MOF solution to respectively obtain MoS 2 @ GO activation solution and Ru-MOF activation solution;
(3) Adding a 5-FU antibody solution into the Ru-MOF activation solution, and reacting to obtain a Ru-MOF/Ab probe;
(4) The MoS is processed 2 The @ GO activation solution is dripped on the surface of a glassy carbon electrode, and after drying, the 5-FU envelope antigen solution is dripped for incubation to obtain MoS 2 @ GO/OVA substrates;
dropping the Ru-MOF/Ab probes to the MoS 2 And reacting on a @ GO/OVA substrate to obtain the electrochemiluminescence immunosensor.
2. The use of claim 1, wherein in step (1), the reaction is carried out at a temperature of 70-90 ℃ for a time of 20-30h.
3. The use according to claim 1, wherein in step (1), the heating is carried out under the conditions of 40-60 ℃ for 0.8-1.2h and then the temperature is increased to 180-220 ℃ for 20-28h.
4. Use according to claim 1, wherein in step (1), (NH) 4 ) 6 Mo 7 O 24 And thiourea in a molar ratio of 1:16-25.
5. The use of claim 1, wherein in step (2), the activation is carried out by adding NHS solution and EDC solution, and the activation time is 1-2h; the concentration of the NHS solution and the EDC solution is 7-9mg/mL.
6. The use of claim 1, wherein in step (3), the volume ratio of the Ru-MOF activating solution to the 5-FU antibody solution is 1:0.5-2.
7. The use of claim 1, wherein in step (3), the Ru-MOF activating solution and the 5-FU antibody solution are reacted and then BSA solution is added.
8. Use according to claim 1, wherein in step (4), moS 2 The mass ratio of the @ GO to the 5-FU coating antigen is 1:15-25.
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